This disclosure relates generally to methods for determining the presence of creatine kinase isoenzymes in a sample. More specifically, this disclosure relates to a method for determining, either qualitatively or quantitatively, the presence of creatine kinase isoenzymes in a sample via immobilized antibody-isoenzyme complexes.
It was demonstrated in 1964 that human tissues contain three principal isoenzymes of creatine kinase (CK, E.C. 2.7.3.2, also known as creatine-ATP-transphosphorylase) which are composed of two unique subunits, M and B, in different combinations. See, e.g., A. Burger et al., Biochem. Z, 339, 305 (1964); D. H. Deul and J. F. L. van Bremen, Clin. Chim. Acta, 10, 276 (1964); and K. Sjovall and A. Voight, Nature, 202, 701 (1964). Thus, such isoenzymes consist of an anodal isoenzyme (BB) present in brain, an isoenzyme (MM) with electrophoretic mobility corresponding to .gamma.-globulin present in skeletal muscle, and an isoenzyme (MB) of intermediate electrophoretic mobility present in some skeletal muscles and in myocardium.
During the next ten years, researchers studied the separation and quantitation of CK isoenzymes, primarily by electrophoretic techniques, and proposed that the MB isoenzyme provides a sensitive and specific indication of acute myocardial infarction. See, for example, A. F. Smith, Clin. Chim. Acta, 39, 351 (1972); H. Somer and A. Konttinen, Clin. Chim. Acta, 36, 531 (1972); and G. S. Wagner et al., Circulation 47, 263 (1973). The separation of CK isoenzymes also was achieved by ion-exchange column chromatography. D. W. Mercer, Clin. Chem., 20, 36 (1974).
More recently, other, perhaps less traditional, methods have been applied to the separation and/or quantitation of the CK isoenzymes. One such method is immunotitration or immunoinhibition. According to E. Jockers-Wretou and G. Pfleiderer, Clin. Chim. Acta, 58, 223 (1975), antisera against the crystallized CK isoenzymes from human skeletal muscle (MM) and from human brain (BB) were produced in rabbits. Both the MM and BB isoenzymes were quantitatively precipitated by their homologous antisera, with no cross-reaction being observed. The hybrid MB isoenzyme from human heart muscle could not be precipitated completely by either of the two antisera. The concentrations of the three creatine kinase isoenzymes in artificial mixtures were then determined from the percentage of non-precipitable activity in the supernatant after reaction with each of the antisera. Because about ten percent residual activity always remained in the supernatant, even with a large excess of antiserum present, it was assumed that such activity resulted from the presence of MB. Consequently, the calculations leading to a value for MB activity were based upon such assumption.
An immunological method very similar to the above is that of D. Neumeier et al., Klin. Wochenschr., 53, 329 (1975); see also U.S. Pat. No. 4,067,775 to U. Wurzburg et al. These workers report that MM and MB are quantitatively precipitated by the MM antiserum. Similarly, BB and MB are quantitatively precipitated by the BB antiserum. Furthermore, BB reportedly is not present in the serum. Consequently, the method involves first determining total CK activity. Activity remaining in the supernatant after precipitation with BB antiserum then is measured. The difference between the two values allegedly represents MB activity in the serum sample.
Radioimmunoassays for the CK isoenzymes also are known. According to D. Neumeier et al., Clin. Chim. Acta, 79, 107 (1977), the subunit B in MB and BB is quantitated by means of a double-antibody radioimmunoassay which utilizes .sup.125 I-labeled BB antiserum. A competitive displacement radioimmunoassay for MB, utilizing .sup.125 I-labeled CK isoenzymes, was reported by R. Roberts et al., Clin. Chim. Acta, 83, 141 (1978). Finally, double-antibody radioimmunoassays for MM and BB using .sup.125 I-labeled isoenzymes were reported by A. C. Van Steirteghem et al., Clin. Chem., 24, 414 (1978) and M. H. Zweig et al., Clin. Chem., 24, 422 (1978), respectively.
Finally, an immunoadsorbent procedure for quantitating CK isoenzymes was reported by J. Perriard et al., Arch. Biochem. Biophys., 191 90, (1978). The procedure involved preparing pure MM and BB antisera and separately coupling such antisera to cyanogen bromide-activated Sepharose 4B. The total CK activity in a sample then was determined. Aliquots of the sample were passed over each immunoadsorbent column and unbound isoenzyme was washed off each column. Unbound isoenzyme activity then was measured in each case. Thus, unbound activity from the anti-MM column represented BB activity and MM activity was the unbound activity from the anti-BB column. MB activity then was determined by subtracting the MM and BB activities from the total CK activity in the sample.
In all of these prior art methods, isoenzyme activity of the isoenzyme-antibody complexes was either presumed or known to be nonexistant. This, of course, is consistent with the known CK isoenzyme antibody inactivation. See, for example, C. A. Williams and M. W. Chase, Editors, "Methods in Immunology and Immunochemistry", Volume IV, Academic Press, New York, 1977, p. 317; B. Cinader, Editor, "Antibodies to Biologically Active Molecules", Volume 1, Proceedings of the 2nd Meeting of the Federation of European Biochemical Societies, Vienna, 21-24 April 1965, Pergamon Press, Symposium Publications Division, Oxford, 1967, p. 87; B. Cinader, Ann. N. Y. Acad. Sci., 103(2), 500 (1963); and M. R. J. Salton, Editor, "Immunochemistry of Enzymes and Their Antibodies", John Wiley & Sons, New York, 1977, p. 104.
Thus, no reports are known in which the presence of CK isoemzymes was demonstrated by measuring the CK isoenzyme activity of an antibody-isoenzyme complex.